Nucleotide sequence and analysis of the Vibrio alginolyticus sucrose uptake-encoding region

The nucleotide sequence of the Vibrio alginolyticus sucrose uptake-encoding region was determined, and contained two genes, scrA and scrK. The scrA gene encodes an enzyme IISucrose (EIIScr) protein of the phosphoenolpyruvate dependent phosphotransferase system and the scrK gene encodes a fructokinase. The deduced amino acid (aa) sequence for the V. alginolyticus EIIScr protein was homologous with the EIIScr proteins from Streptococcus mutans, Salmonella typhimurium (pUR400 system) and Bacillus subtilis. The deduced aa sequence for the V. alginolyticus fructokinase was homologous with the Escherichia coli enzymes, 6-phosphofructokinase (isoenzyme 2) and ribokinase. Transposon phoA mutagenesis experiments indicated that the EIIScr protein was a membrane-bound protein with a region that extended into the periplasm.     

Deletion analysis of sucrose metabolic genes from a Salmonella plasmid cloned in Escherichia coli K12

The sucrose operon from pUR400, a 78-kbp conjugative Salmonella plasmid, was cloned in Escherichia coli K12. The operon was located in a 5.7-kbp SalI restriction fragment and was subcloned, in each of two possible orientations, using the expression vector pUC18. The insert DNA was restriction mapped and duplicate restriction sites in the insert and in the polylinker of the vector were used to create various deletions promoter distal in the operon sequence. Additional deletions were made with the restriction exonuclease Bal31. Cells containing hybrid plasmids with specified deletions lacked the ability to transport sucrose or were constitutive for hydrolase and/or uptake activities. The scrA (enzyme IIScr) and scrR (regulatory) genes resided within 2900-bp SmaI-SalI DNA fragment and were assigned the order scrB, scrA, scrR. An amplified sucrose-inducible gene product, Mr 68,000, was detected only in the membrane fraction from recombinant cells that contained plasmid with the intact operon sequence. This protein represented 11% of the total membrane protein and was resistant to extraction with 0.5 M sodium chloride, 2% Triton X-100, and 0.5% sodium deoxycholate. The protein did not appear to be the product of either the scrA, scrB, or scrR gene and may therefore represent a previously unidentified membrane-bound sucrose protein. A new gene, scrC, is proposed. In addition, the cloned 5.7-kbp SalI and 2.5-kbp SmaI-SalI DNA fragments failed to hybridize to chromosomal DNA from Bacillus subtilis, Streptococcus lactis, Streptococcus mutans, and Lactobacillus acidophilus as well as to DNA from a sucrose plasmid from Salmonella tennessee. However, the probes showed weak homology with a 20-kbp EcoRI restriction fragment from Klebsiella pneumoniae.     

Utilization of a mini-mu transposon to construct defined mutants in Streptococcus mutans

The gtfB gene coding for glucosyltransferase-I (GTF-I) activity previously isolated from Streptococcus mutans GS-5 was insertionally inactivated with the newly constructed transposon MudE in an Escherichia coli background. Insertion of MudE into various regions of the gtfB gene led to inactivation of GTF-I activity. The altered gene was introduced back into S. mutans GS-5 by transformation and produced mutants defective in insoluble glucan synthesis as well as the ability to colonize smooth surfaces in the presence of sucrose. Therefore, the MudE transposon can be utilized to produce specific mutants in oral streptococci as well as in other transformable Gram-positive bacteria expressing an erythromycin-resistance marker.     

Overlapping substrate specificity for sucrose and maltose of two binding protein-dependent sugar uptake systems in Streptococcus mutans

Sugar metabolism by Streptococcus mutans is associated with tooth decay. The most abundant sugars in the human diet are sucrose and maltose, a derivative of starch. Previously, we reported a binding protein-dependent transport system (msm) in S. mutans that transports sucrose and maltose, but its associated enzymes do not metabolize maltose. By searching the S. mutans genomic sequence for a maltose system (mal), we found a gene cluster encoding proteins with homology to those of msm and the Escherichia coli maltose system. Mutants were constructed by deleting msm or mal, or both, and tested for sugar utilization. Deletion of the mal system diminished the ability of S. mutans to ferment maltose, but deletion of only the mal transporter genes or msm showed reduced utilization of chromogenic maltosides. Maltose, sucrose, glucose, fructose, mannose, and N-acetyl glucosamine inhibited utilization of chromogenic maltosides by the wild-type strain and mutants. In conclusion, the two binding protein-dependent systems in S. mutans appear to transport collaboratively their common substrate sugars, notably sucrose and maltose.     

Multiple Streptococcus mutans Genes Are Involved in Biofilm Formation

Streptococcus mutans has been strongly implicated as the principal etiological agent in dental caries. One of the important virulence properties of these organisms is their ability to form biofilms known as dental plaque on tooth surfaces. Since the roles of sucrose and glucosyltransferases in S. mutans biofilm formation have been well documented, we focused our attention on sucrose-independent factors. We have initially identified several mutants that appear to be defective in biofilm formation on abiotic surfaces by an insertional inactivation mutagenesis strategy applied to S. mutans. A total of 27 biofilm-defective mutants were isolated and analyzed in this study. From these mutants, three genes were identified. One of the mutants was defective in the Bacillus subtilis lytR homologue. Another of the biofilm-defective mutants isolated was a yulF homologue, which encodes a hypothetical protein of B. subtilis whose function in biofilm formation is unknown. The vast majority of the mutants were defective in the comB gene required for competence. We therefore have constructed and examined comACDE null mutants. These mutants were also found to be attenuated in biofilm formation. Biofilm formation by several other regulatory gene mutants were also characterized using an in vitro biofilm-forming assay. These results suggest that competence genes as well as the sgp and dgk genes may play important roles in S. mutans biofilm formation.     

Carbon catabolite repression of sucrose utilization in Staphylococcus xylosus: catabolite control protein CcpA ensures glucose preference and autoregulatory limitation of sucrose utilization

Sucrose utilization in Staphylococcus xylosus is dependent on two genes, scrA and scrB; encoding a PTS permease and a sucrose phosphate hydrolase, respectively. The genes are encoded on separate loci and are transcribed from two promoters, P(scrA) and P(scrB), both of which are controlled by the repressor ScrR by binding to the operator sequences O(A) and O(B). In the scrA promoter region, a catabolite-responsive element (cre), operator for the global catabolite control protein CcpA, is also present, but its contribution to scrA regulation has not been determined. Using an integrative promoter probe plasmid, the activities of the promoters P(scrA) and P(scrB) were determined under different growth conditions. Both promoters are induced by sucrose and induction is prevented when glucose is also present. Without a functional CcpA, glucose-mediated prevention of induction is lost, clearly demonstrating that CcpA ensures hierarchical sugar utilization with glucose as preferred substrate. Measurements of promoter activities in the absence of a functional ScrR repressor indicated that CcpA also acts upon the operators O(A) and O(B), albeit not as efficiently as on the genuine cre in P(srcA). Besides determining the choice of the carbon source, CcpA has a second effect on sucrose gene expression. When sucrose is the sole carbon source, sucrose catabolism activates carbon catabolite repression and CcpA prevents full induction of the sucrose utilization genes by partially repressing the scrA promoter. Thus, CcpA-dependent regulation serves as a built-in autoregulatory device to restrict sucrose uptake.     

Phosphoenolpyruvate-dependent phosphotransferase system enzyme III and plasmid-encoded sucrose transport in Escherichia coli K-12.

The phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system enzyme IISCR, specific for and regulated by sucrose, was analyzed in derivatives of Escherichia coli K-12 carrying the sucrose plasmid pUR404. Enzyme IIScr, coded for by gene scrA of the plasmid, depended for its transport and phosphorylation activity directly on the phosphotransferase system enzyme IIIGlc, Scr, coded for by the chromosomal gene crr.     

Molecular structure of the locus for sucrose utilization by Lactobacillus plantarum: comparison with Pediococcus pentosaceus

Structure of the sucrose utilization locus in a Lactobacillus plantarum type strain was studied using PCR and Southern hybridization. Restriction map analysis revealed its high similarity to the sequenced sucrose utilization locus of Pediococcus pentosaceus pSRQ1. The L. plantarum locus proved containing oppositely oriented scrA and the scrBRagl operon, but not agaS. The L. plantarum sucrase gene (scrB) was partly sequenced. A higher (98.6%) homology was revealed between scrB than between the 16S rRNA genes of L. plantarum and P. pentosaceus, suggesting horizontal transfer of the sucrose utilization locus between the genera of lactic acid bacteria. Amino acid sequence analysis showed that the ScrB proteins of the two species belong to a subfamily of glycosyl hydrolase family GH32 which includes various beta-fructosidases.     

The role of glucan-binding proteins in the cariogenicity of Streptococcus mutans

Streptococcus mutans produces glucan-binding proteins (Gbps), which appear to contribute to the virulence of S. mutans. GbpA and GbpC genes were inactivated by the insertion of antibiotic-resistant genes into each gbp gene of S. mutans MT8148 to generate Gbp-defective mutants. Sucrose dependent adherences of the GbpA- and GbpC-defective mutants were found to be significantly lower than those of their parent strains MT8148. Caries inducing activity of the mutants in rats was significantly lower than that of strain MT8148R (streptomycin-resistant strain of MT8148). These results suggest that GbpA and GbpC participate in cellular adherence to tooth surfaces and contribute to the cariogenicity of S. mutans.     

Use of a novel mobilizable vector to inactivate the scrA gene of Streptococcus sobrinus by allelic replacement.

The virulence factors of the cariogenic bacterium Streptococcus sobrinus have been difficult to assess because of a lack of tools for the genetic manipulation of this organism. The construction of an Escherichia coli-Streptococcus shuttle vector, pDL289, that can be mobilized into S. sobrinus by the conjugative plasmid pAM beta 1 was described in a previous report. The vector contains pVA380-1 for replication and mobilization in streptococci, the pSC101 replicon for maintenance in E. coli, a kanamycin resistance marker that functions in both hosts, and the multiple cloning site and lacZ from pGEM7Zf(-). pDL289 is stable with or without selection in several species of Streptococcus. In this study, a derivative with a deletion in the minus origin of the pVA380-1 component of pDL289 was constructed. This derivative, pDL289 delta 202, was less stable than pDL289 in Streptococcus gordonii Challis, Streptococcus mutans, and S. sobrinus. Both pDL289 and pDL289 delta 202 were mobilizable by pAM beta 1 into S. sobrinus, with frequencies of 3 x 10(-6) and 1 x 10(-7) transconjugants per recipient CFU, respectively. The cloned scrA gene of S. sobrinus 6715-10 coding for the EIISuc of the sucrose-specific phosphoenolpyruvate phosphotransferase system was interrupted by the insertion of a streptococcal spectinomycin resistance gene active in E. coli and streptococci. The interrupted scrA gene was subcloned into both pDL289 and pDL289 delta 202. Each recombinant plasmid was introduced into the DL1 strain of S. gordonii Challis, which was then used as a recipient for the conjugative transfer of pAM beta 1. The latter plasmid was used to mobilize each recombinant plasmid from S. gordonii Challis DL1 to S. sobrinus 6715-10RF. Subsequently, recombinants derived from a double-crossover event were isolated on the basis of resistance to spectinomycin and susceptibility to kanamycin. Recombinational events were confirmed by Southern hybridization, and the inactivation of the EII Suc in double crossovers was confirmed by phosphotransferase system assays. This is the first report of allelic replacement in S. sobrinus.     

Nucleotide sequence of Streptococcus mutans superoxide dismutase gene and isolation of insertion mutants.

A gene (sod) encoding superoxide dismutase (SOD) was cloned from Streptococcus mutans in Escherichia coli, and its nucleotide sequence was determined. The presumptive amino acid sequence of its product revealed that the SOD is basically of Mn type. Insertional inactivation of the sod gene resulted in the loss of SOD activity in crude extracts, indicating that the gene represents the only functional gene for SOD in S. mutans. Moreover, Southern blot analysis indicated that the S. mutans chromosome had no additional gene which was hybridizable with an oligonucleotide probe specific for an SOD motif. The SOD-deficient mutants were able to grow aerobically, albeit more slowly than the parent strains.     

LuxS基因缺陷对变形链球菌生物膜形成的影响

目的观察LuxS基因缺失后变形链球菌生物膜成熟初期的变化情况。方法通过扫描电镜观察标准菌和缺陷菌在不同营养环境中生物膜成熟初期的形成情况。结果对不同营养环境中形成的生物膜观察,发现在富含蔗糖的环境中,缺陷菌成熟初期的生物膜形成能力较标准菌弱。结论 LuxS基因缺失后变形链球菌在蔗糖环境中生物膜形成的能力减弱。     

Expression of a Streptococcus mutans glucosyltransferase gene in Escherichia coli.

Chromosomal DNA from Streptococcus mutans strain UAB90 (serotype c) was cloned into Escherichia coli K-12. The clone bank was screened for any sucrose-hydrolyzing activity by selection for growth on raffinose in the presence of isopropyl-beta-D-thiogalactoside. A clone expressing an S. mutans glucosyltransferase was identified. The S. mutans DNA encoding this enzyme is a 1.73-kilobase fragment cloned into the HindIII site of plasmid pBR322. We designated the gene gtfA. The plasmid-encoded gtfA enzyme, a 55,000-molecular-weight protein, is synthesized at 40% the level of pBR322-encoded beta-lactamase in E. coli minicells. Using sucrose as substrate, the gtfA enzyme catalyzes the formation of fructose and a glucan with an apparent molecular weight of 1,500. We detected the gtfA protein in S. mutans cells with antibody raised against the cloned gtfA enzyme. Immunologically identical gtfA protein appears to be present in S. mutans cells of serotypes c, e, and f, and a cross-reacting protein was made by serotype b cells. Proteins from serotype a, g, and d S. mutans cells did not react with antibody to gtfA enzyme. The gtfA activity was present in the periplasmic space of E. coli clones, since 15% of the total gtfA activity was released by cold osmotic shock and the clones were able to grow on sucrose as sole carbon source.     

Virulence of Streptococcus mutans: biochemical and pathogenic characteristics of mutant isolates.

The in vitro dextran-sucrase activities and adherence to glass of S. mutans 6715 and PS14 wild types and mutants were quantitated and compared with their in vivo cariogenicity in young, gnotobiotic rats. In general, S. mutans PS14 mutants B414 and B421 and 6715 mutant C4 demonstrated less dextran-sucrase activity and adherence than parental strains and caused fewer carious lesions in gnotobiotic rats. Rats monoinfected with either PS14 mutants B414 or B421 had less plaque and viable S. mutans in plaque than rats infected with parental strain. Both S. mutans 6715 mutants C211 and C229, demonstrated greater enzyme activity and adherence than the parental strain and produced more carious lesions.     

Effect of utilization of substrates on the composition of dental plaque

Abstract The microbiota in the mouth is subjected to substrate limitations. In this study we have evaluated the role of competition for carbon and energy substrates on the proportions of 2 microbial species in a simplified plaque ecosystem in gnotobiotic rats. Germ-free rats were inoculated with a combination of Streptococcus sanguis and Streptococcus mutans , or with a combination of Streptococcus milleri and S. mutans . The available carbon and energy sources were varied through the host's diet. 3 Experimental diets were tested: (i) a basal diet low in soluble carbohydrates; (ii) an arginine-supplemented diet; (iii) a sucrose-supplemented diet. Arginine is used for growth by S. sanguis and S. milleri , but not by S. mutans . Sucrose is rapidly fermented by all 3 species.
The total number of viable organisms on the dentition increased when arginine or sucrose were supplied in the diet. With the arginine-supplemented diet, S. sanguis and S. milleri increased while S. mutans decreased. With the sucrose-supplemented diet, S. mutans increased while S. sanguis and S. milleri decreased. These results were explained by assuming that the organism with the highest growth rate on the supplementary substrate competes most favourably. Changes in the environmental pH, due to breakdown of sucrose and arginine, might also have affected the competition between the streptococci. In addition, production of extracellular glucans from sucrose could be a competitive advantage for S. mutans .